Com probe implemented STexS II greatly enhances specificity in SARS-CoV-2 variant detection

The initial introduction of utilizing double helix structural oligonucleotides known as SNP typing with excellent specificity (STexS) in a standard PCR greatly improved the detection of single nucleotide polymorphisms (SNP) by enhancing amplification rates of primer-matching strands and interrupting mismatched strands by constant instability of kinetics regarding alignment attaching and detaching. The model was beneficial overall in detecting SNP variants consisting of large amounts of wildtype strands such as EGFR mutation genotyping for early detection of non-small cell lung cancer. While the STexS PCR is advantageous in detecting SNPs and biomarkers, limitations were yet observed. Despite the ability to detect variants 10 times more effective than a typical amplification-refractory mutation system PCR, it could only perform optimally in DNA concentrations around 101 ~ 105. To further enhance STexS specificity to perform detecting viral-RNA variants such as the infamous SARS-CoV-2, a novel improvement of the regular TaqMan Probe using Com-probes to inhibit high copy wild targets and amplify low copy mutant targets. By introducing the novel STexS II, omicron variants of SARS-CoV-2 were able to be successfully detected in high concentrations of normal genes.


Scientific Reports
| (2023) 13:1036 | https://doi.org/10.1038/s41598-022-24530-w www.nature.com/scientificreports/ after the initial exposure 11 . To cause the SARS-CoV-2 to further diminish, effective detection methods that can discriminate infected strands within yet inflicted strands are essential. To achieve such goals, this study focused on developing the previous STexS method to effectively detect and amplify mutated regions of SARS-CoV-2 while simultaneously diminishing unnecessary strands by inhibiting signal emission to increase specificity. As a result, we introduce an enhanced method utilizing a novel probe mixture named Combination and Competition probes (Com probes).

Results
qPCR enhancements by utilizing Com probes. Unlike cell-based DNA genotyping, viral SNP, especially RNA-based viridae, consists of wide concentrations ranging from 10 1 to 10 8 copies. The typical ARMS PCR was able to detect variants within the 10 1 -10 3 range. STexS was able to improve the detection range to 10 1 -10 5 . Despite the enhanced specificity, viral variants could be seen in far concentrated amounts that exceed the current detection limit. To overcome the challenge and increase specificity, methods such as TaqMan and minor groove binder (MGB) included TaqMan probes were introduced for distinct purposes 12 . While the original Taqman probe focused on normalizing and quantifying relatively small concentrations around 10 1 -10 5 , MGB included TaqMan probes that tend to perform optimally in high concentrations around 10 4 -10 8 . To detect variants of RNA-based viruses such as SARS-CoV-2, the spectrum must be broadened that envelops small and high concentrations 13 . As a result, the concept of STexS II was based on novel TaqMan probe enhancements (Fig. 1).
In the previous research, one of the priorities of optimal signal amplification was adjusting reaction rates and melting temperatures (Tm) to be above the lowest annealing temperature to rule out unnecessary dimers 7 . Similarly, the correlation between probe Tm and PCR signal elevation needed to be observed. By targeting the main mutation sites of the omicron variant Q954H, T547K, and delta variant T19R, the overall PCR signal was correlated with probe Tm increase (Fig. 2). Further elongating the probe sequence resulted in a gradual increase of an average 3 °C per base alongside a higher increase in mutant amplification. Despite the increased amplification rates, the increase was also observed in wildtype strands leading to a decrease in cycle threshold (Ct) value when quantified (Sup Table 1). To block unwanted signals from amplifying, the probe sequence alone needed to be shortened, forfeiting the advantages of having high specificity in the detection of low-copy targets (Table 1). To use an extended probe and block WT signals, a different approach was inevitable.
Combining several probes within a single PCR is not a novel concept. A typical probe combination consists of probes emitting different colors which attach to different primers 14 . This condenses the PCR workflow to quantify several targets simultaneously. The multiplex method of probes was possible as probes were designed to react independently with no interference within each group 15 . While this is the main approach for using multiplex probes, the STexS II Com probes were implemented to interfere with and block nonspecific signals, gradually increasing the gap between mutated and nonspecific signals. Each Com probe was designed to attach wildtype regions that parallel to the mutated sequence ( Table 2). For instance, the FAM-Q954H probe and its Com probe target the identical sequence of the Q954H region. While the Q954H probe targets the mutated www.nature.com/scientificreports/ sequence, the Com probe of Q954H targets the unaltered sequence. Once the probes are activated, the mutated target signals will be attached to target probes and initiate amplification, while the nonspecific strand will compete in its binding phase between the Q954H and Com probe. Initially, the improved STexS II method was tested whether it improves the previous dynamic range. As a result, STexS II successfully detected low (10 2 ) and high (10 8 ) concentrations (Sup Fig. 1). After validation, surprisingly, Com probes drastically decreased the amplification of nonspecific signals of Q954H, T547K, and T19R regions (Fig. 3). Because the Com probe has a similar structure against the target probe, further analysis for finding signs of probe interference was performed. When the Ct value of each target was measured, Com probes did not hinder the overall quantification in a significant manner (Sup Table 2). As a result, mutation detection of the omicron variant was successful using probes in a combinative and competitive concept.  www.nature.com/scientificreports/ For further validation and optimization, multiple trials regarding STexS II were performed adjusting the amount of probes and probe sequences. Analyzing multiple trials of PCR targeting Q954H, probes that are elongated amplified a higher Ct threshold compared to the original template (Fig. 4a). The overall enhanced specificity did affect the increase in target amplification. When accompanied by Com probes, the signal of nonspecific strands was mostly diminished, resulting in mostly positive detections (Fig. 4b). Further increasing the usage of probes and Com probes increased the overall signal threshold, but since the previous Ct value was high enough for positive results, even a small volume of Com probes was sufficient for effective PCR detection.
Once the potential of Com probes was observed, RNA samples of omicron variants were further tested whether the novel STexS II will be effective in lower copies. Three trials of PCR targeting the omicron mutation site Q954H and T547K were performed with identical conditions applied in Fig. 4 (Sup Table 3). When   www.nature.com/scientificreports/ comparing PCR detection with or without the use of Com probes in high copies of 2 × 10 4 , all methods were able to successfully result in positive detection (Fig. 5). However, when the RNA copy was decreased, only the PCR using Com probes with elongated probes was able to yield positive detection due to increased specificity alongside diminished amplification of nonspecific strands. The results confirm that STexS II can perform not only in high concentrations of RNA but also in low densities.

Discussion
For PCR genotyping to successfully detect mutation, not only the ability to efficiently bind to the target region is crucial, but also the difference between target strands and nonspecific strands needs to be significant to consider as a positive result. Hence the detection will still be viable even though the actual Ct value of targets would be low due to diminished amplification rates of nonspecific strands. Typical PCR methods overcome such obstacles by using short probes to block any unwanted binds. However, using relatively short probes would lead to two major disadvantages: the low specificity due to low Tm and low amplification rates of target strands not having a stable bind during alignments. The low amplification rate will eventually mean the method will not be viable in low concentrations and RNA targets such as SARS-CoV-2 detection will be a challenge. Substituting probes with longer sequences indeed increases the amplification of targeted templates but companies with an increase of WT signals. Despite the probe and WT having mismatches, a small portion of the abnormal bond proceeds degradation resulting in a traceable signal hindering the overall Ct value. The introduced STexS II overcomes the problem by implementing an elongated main probe that effectively amplifies the target strand and a secondary probe that competes with the main probe in nonspecific binding. The normal concept of probes is to successfully match to templates and emit detectable signals through fluorescence. However, Com probes shift the approach by intentionally binding to WT strands without hydrolyzable dyes. As the Com probe perfectly matches with the WT strand, no further displacements will occur once it is matched, further decreasing the main probe to have mismatches. When the Com probe couples with WT strands, the actual amplification cannot be quantified since no fluorescence is detected after degradation (Fig. 6). Therefore, the STexS II platform can effectively yield positive detection by increasing the gap between high specific target amplification and untraceable nonspecific signals. The method further discriminates the target region of the omicron variant in both high and low concentrations. www.nature.com/scientificreports/ By achieving such an effective method, the STexS II can be implemented on various occasions. Whether it would be a DNA or RNA environment, high or low concentrated copy, using the novel concept of combining probes in a competitive manner will successfully perform in detecting mutation. Also despite the constant mutation leading to a novel SARS-CoV-2 variant inevitable, STexS II will continue to provide an effective method to further detect mutations and overall understanding for future diagnosis.

Materials and methods
All methods were performed and approved under the supervision and guidelines of the Korean Research Institute of Bioscience and Biotechnology (KRIBB) and the Korean National Culture Collection for Pathogens (NCCP). Both institutes are government affiliations that confirm integrity and regulations.
Primer and probe design. Each primer was designed and manufactured to match the omicron variant mutation location Q954H and T547K. Probes were designed and manufactured to match above mentioned primers. Probe elongation was performed by adding one base to the 3' end. A Series of probes elongation was tested for optimal performance and Tm adjustments. Com probes were designed to not emit any fluorescent color only to act as a competitive combination in binding nonspecific probes. The sequence of Com probes was designed to be one base different from the main probe. Omicron variant standard sample. RNA samples of omicron variants of SARS-CoV-2 were obtained publicly through the National Culture Collection for Pathogens (NCCP). Three sample collections NCCP43326, NCCP43410, and NCCP43408 were stored for further STexS II trials.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.